Human Reproduction, Vol.24, No.9 pp. 2205– 2216, 2009 Advanced Access publication on May 21, 2009 doi:10.1093/humrep/dep187
ORIGINAL ARTICLE Reproductive biology
Differential expression of calreticulin, a reticuloplasmin in primate endometrium T. Parmar 1, S. Nimbkar-Joshi 1, R.R. Katkam 1, S. Gadkar-Sable 1, U. Chaudhari 1, D.D. Manjramkar 2, L. Savardekar 3, S. Jacob 1, C.P. Puri 1, and G. Sachdeva 1,4 1 Primate Biology Division, National Institute for Research in Reproductive Health, Indian Council of Medical Research, Parel, Mumbai 400012, India 2Experimental Animal Facilities, National Institute for Research in Reproductive Health, Indian Council of Medical Research, Parel, Mumbai 400012, India 3Clinical Research Division, National Institute for Research in Reproductive Health, Indian Council of Medical Research, Parel, Mumbai 400012, India 4
Correspondence address. Tel: þ91-22-24192111; Fax: þ91-22-24139412; E-mail:
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background: To our knowledge, there are no data on hormonal regulation of reticuloplasmins in primate endometrium. We report the presence and modulation of expression of three reticuloplasmins in endometrium of bonnet monkeys (Macaca radiata). methods: Receptive and non-receptive endometria obtained from vehicle-treated control and onapristone (antiprogestin)-treated animals, respectively, were compared for differentially expressed proteins by two-dimensional proteomics. Mass spectrometric analysis annotated two such proteins as calreticulin and protein disulfide-isomerase (PDI), known to be molecular chaperones in endoplasmic reticulum. We then investigated if endoplasmin, another reticuloplasmin is also differentially expressed. Expression of these reticuloplasmins was also investigated in the endometriuma during pregnancy in bonnet monkeys. Samples were analysed by immunohistochemistry and western blot (calreticulin in human endometrium), and calreticulin transcript levels in Ishikawa cell line were assessed by real time PCR.
results: Immunohistochemical analysis of the functionalis region of non-receptive endometria in monkeys revealed higher expression of (i) calreticulin (P , 0.01) in glandular epithelium and (ii) PDI in stroma (P , 0.0001), but no change in endoplasmin in stroma or glands, compared with receptive endometria. Protein level of all three reticuloplasmins in the stromal region of endometrial functionalis was higher in pregnant than non-pregnant animals (P , 0.05). Human endometrial calreticulin protein was higher in the estrogen-dominant (proliferative) phase than progesterone-dominant (mid-secretory) phase of the cycle. Calreticulin mRNA in Ishikawa cells is up-regulated by estrogen (P , 0.05 versus control), with a trend towards down-regulation by progesterone.
conclusion: Our data suggest that endometrial reticuloplasmins are regulated by hormones and embryonic stimuli in a cell-type specific manner. These novel data open up new lines of investigation for elucidating the mechanisms by which hormones or embryonic stimuli influence the sub-cellular physiology of endometrium. Key words: endometrium / receptivity / reticuloplasmins / calreticulin / protein disulfide-isomerase
Introduction Role of ovarian hormones in the acquisition of distinct structural and molecular signatures by the endometrium is indisputable. Specific molecular patterns are achieved by endometrium during the midsecretory or progesterone dominant phase of menstrual cycle. These molecular prints act as signposts of the receptivity for an incoming embryo. However, the mechanisms by which progesterone contributes to endometrial receptivity are largely unknown in primates. This may be partly attributed to the lack of composite
information on the endometrial factors that are progesterone regulated. Recently several microarray-based investigations have been undertaken to generate transcriptomes of human endometrium in the progesterone dominant phase (Carson et al., 2002; Kao et al., 2002; Borthwick et al., 2003; Riesewijk et al., 2003; Ponnampalam et al., 2004; Talbi et al., 2006). However, it is being increasingly realized that these data need to be complemented with high throughput protein profiling using proteomics approaches. The present study was initiated using a comparative two-dimensional (2D) proteomics approach to identify progesterone-regulated proteins
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2206 in the mid-secretory phase endometrium of bonnet monkeys (Macaca radiata). To identify these proteins, animals were treated with a select dose of an antiprogestin-onapristone. Onapristone prevents the binding of progesterone to its receptors (Horne and Blithe, 2007) and renders endometrium refractory to progesterone action. Our previous studies demonstrated that onapristone at selected dosages does not adversely affect the menstrual cycle length, ovulation and peripheral levels of estradiol (E2) and progesterone (Katkam et al., 1995) but renders endometrium implantation-incompetent or non-receptive. Our previous studies also revealed differential expression of endometrial integrins, leukemia inhibitory factor, transforming growth factor b2 and its receptor and other factors such as Rab coupling protein at transcriptional and/or translational level in onapristone treated animals (Puri et al., 2001; Sachdeva et al., 2001; Patil et al., 2005). The present study employed comparative 2D proteomics of endometrial tissues to identify differentially expressed proteins in onapristone- and vehicle-treated animals. Two differentially expressed proteins were annotated as calreticulin and protein disulfide isomerase (PDI). Interestingly both these proteins are known for their similarity in terms of (a) sub-cellular localization (b) regulatory elements in promoter regions and (c) functions such as protein folding and calcium homeostasis. Expression of these proteins was investigated in the endometrial functionalis during two contrasting conditions i.e. one (curtailment of progesterone action) which rendered endometrium non-receptive and another, wherein not only progesterone action was optimal but embryonic stimuli were also operative. Studies were also carried out to investigate whether the expression of endometrial calreticulin is regulated by hormones in vitro. To our knowledge, hitherto there are no data on the presence of these reticuloplasmins in primate endometrium and their regulation by steroids or embryonic stimuli.
Materials and Methods The study was approved by the Animal Ethics Committee of the Institute and the Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Social Justice and Empowerment, Government of India. The study was also approved by the Institute’s ethics committee for collection of samples from healthy women.
Bonnet monkeys (Macaca radiata) Adult bonnet monkeys were housed singly under controlled conditions in the animal house facility of the Institute. Animals were fed with a diet composed of semi-formulated Indian bread, fresh seasonal fruits, eggs and sterile water. Female bonnet monkeys, each weighing between 3.5 and 4.5 kg and showing at least two consecutive ovulatory menstrual cycles of 28– 30 days were included in the study. Cyclicity of the animals was monitored by vaginal swab examination. Serum E2 and progesterone levels in regularly cycling animals were measured by specific radioimmunoassays as described previously (Patil et al., 2005). Female bonnet monkeys with normal hormonal profiles (peak levels-E2: 300 – 600 pg/ml; progesterone: 3 – 6 ng/ml) were included in the study.
Study 1: induction of endometrial non-receptivity by antiprogestin treatment Onapristone-ZK 98.299 (a kind gift from Dr. Walter Elger, Schering, Germany), an antiprogestin, dissolved in vehicle (benzyl benzoate: castor
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oil, 9:1), was injected s.c. at a dose of 5.0 mg into the animals on day 1 of their menstrual cycle and continued every third day for one cycle until the day of biopsy. This treatment regimen was efficacious in inhibiting implantation in bonnet monkeys (Katkam et al., 1995). Endometrial biopsies were collected on day 8 post-E2 peak from vehicle-treated control (n ¼ 3) and onapristone treated (n ¼ 3) animals.
Study 2: superimposition of endometrial receptivity by embryonic stimuli Regularly cycling female bonnet monkeys with normal hormonal profiles were mated with male of proven fertility for six continuous days starting from 2 days prior to the expected E2 peak. Pregnancy was detected using the preimplantation factor (PIF) bioassay (Rosario et al., 2005a, b, 2008). In brief, sera were collected from animals on day 6 post-E2 peak. Heat inactivated serum was incubated with a mixture of lymphocytes and platelets obtained from blood of ‘O’ Rh-positive male donors. This mixture was further incubated with anti-CD2 antibody and rabbit complement (Sigma-Aldrich, USA). Lymphocyte-platelet rosettes were counted. The reaction was considered as positive if more than 9% rosettes were observed (Coulam et al., 1995; Roussev et al., 1995). The bioassay has been extensively validated for its suitability as a marker of early pregnancy (Rosario et al., 2005a). PIFpositive animals were termed as pregnant while PIF negative animals were termed non-pregnant. Uteri were removed on day 9 post-E2 peak (the day of initiation of implantation) from pregnant (n ¼ 3) and nonpregnant (n ¼ 3) animals. These samples were fixed in 4% buffered formalin and embedded in paraffin. Paraffin blocks were sectioned at 5 mm and mounted on 0.1% poly-L-lysine coated slides for histological and immunohistochemical analysis. Embryo attachment site was observed as a hematose spot in the fundal region of uterus in PIF positive animals on day 9 post-E2 peak. This reaffirmed the utility of PIF in detecting early pregnancy. Immunostaining was also carried out using antibodies against OCT-4 to identify embryonic cells at the attachment site (data not shown). Endometria from the uterine pole which showed the presence of embryonic cells at morphological and immunohistochemical level will be hereafter referred to as the implantation pole endometrium (IPE), and that from the opposite pole as the non-implantation pole endometrium (NIPE). Endometria from non-pregnant animals will be referred to as nongestational endometrium (NGE).
Human participants Regularly cycling women (21– 35 years) of proven fertility with a history of at least one live birth were enrolled in the study. The menstrual cycle length in these women was 28 + 2 days (mean + SD). Women using any hormonal contraceptive methods and women with signs or symptoms of lower genital tract infections or with polycystic ovary syndrome, uterine fibroids or luteal phase defect were excluded. All women gave informed consent to participate in the study. Ovulation was monitored by serial ultrasonography (USG) to ascertain the follicular collapse. Endometrial tissue samples were collected using a probet from six women on day 6 post-ovulation (i.e. mid-secretory phase) and from five women on day 2 – 3 prior to ovulation (i.e. proliferative phase) as described previously (Parmar et al., 2008). The first USG was performed on day 6 or 7 of the menstrual cycle, depending on length of the last menstrual cycle, the second USG on day 8 or day 9 and then daily until follicle rupture, i.e. ovulation, was evident. Proliferative phase samples were collected on day 2 – 3 prior to the expected day of ovulation, predicted from length of the last menstrual cycle. USG was continued in these subjects until follicle rupture was observed. This facilitated retrospective determination of the actual day of sample collection with respect to the day of ovulation. Blood samples were also collected to
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determine serum levels of progesterone on the day of biopsy collection and thereby ascertain luteal sufficiency.
Ishikawa cell line The Ishikawa endometrial cell line derived from a human endometrial adenocarcinoma was purchased from Sigma-Aldrich (Catalogue no: 99040201). Ishikawa cells are estrogen receptor and progesterone receptor positive (Castelbaum et al., 1997). The cell line was maintained in phenol red free Dulbecco’s modified Eagle’s medium (DMEMþ) Ham’s F12 (1:1, vol/vol) supplemented with fetal calf serum (FCS) at 95% air-5% CO2 in humidified chambers at 378C. Cells (2 106) were seeded in T75 culture flask 16 h prior to hormone stimulation studies. The cells (at 50 – 60% confluency) were then treated with phenol red free DMEMþ Ham’s F12 (1:1, vol/vol) containing 5% charcoal stripped FCS with or without steroids. Hormone stocks (1000-fold concentrated) were prepared in 100% ethanol. E2 (1028 mol/l, final concentration) or progesterone (1026 mol/l, final concentration) or vehicle (0.001% ethanol) was added to cells as described elsewhere (Parmar et al., 2008). Cells were harvested after 2 days for real-time PCR and western blot analysis to determine calreticulin expression. These experiments were repeated at least three times.
Histological analysis Endometrial tissues fixed in 10% buffered formalin were washed in 70% vol/vol ethanol for 24 h and embedded in paraffin. Paraffin sections were cut at 5 mm and stained with hematoxylin/eosin. Endometrial biopsies were dated according to Noyes’ criteria (Noyes et al., 1950). Histological features of endometrial samples matched well with the features characteristic of the phase during which samples were collected.
Two-dimensional gel electrophoresis and protein identification Endometrial tissue (100 mg) suspended in 1.0 ml of lysis buffer (9 M urea, 4% (3-[(3-Cholamidopropyl)-Dimethylammonio]-1-Propane Sulfonate) (CHAPS), 40 mM Tris) was homogenized using IKA T25 ultra-turrax homogenizer (Cole-Parmer, Illinois, USA) on ice. The homogenate was centrifuged at 18 921 g for 30 min at 48C. Proteins were extracted from supernatants using 2D clean up kits as per the manufacturer’s instructions (Amersham Pharmacia Biotech, Uppasala, Sweden). The protein rich pellets were solubilized in rehydration buffer containing 9 M urea, 10 mM dithiothreitol (DTT), and 0.5% CHAPS. Protein concentration was determined using the Bradford assay (Bradford, 1976). Immobiline pH gradient strips 11 cm long (in the non-linear range of pH 3 – 11 or in the linear range of pH 4 – 7) were actively rehydrated with protein sample at 50 V for 12 h at 208C. Isoelectric focusing (IEF) was carried out in an IEF cell (BioRad, Richmond, CA, USA) for either 30 000 V h (for 11 cm strips) or 20 000 V h (for 7 cm strips). The strips were then reduced and then alkylated in buffer containing 50 mM Tris – HCl (pH 8.8), 6 M urea, 30% glycerol, 65 mM DTT (for reduction) or 135 mM iodoacetamide (for alkylation), 2% sodium dodecyl sulphate (SDS) for 30 min each at room temperature. Protein resolution in the second dimension was carried out using 10% SDS-polyacrylamide gel electrophoresis (PAGE) gels. Polypeptide spots on 2D gels were visualized by a mass spectrometric compatible silver staining method (Mortz et al., 2001). Each sample was run at least three times to ensure reproducibility. 2D gel images were acquired using Imagescanner (Amersham Biosciences, Uppsala, Sweden) and analysed using Image Master 2D Platinum software version 5.0 (Amersham Biosciences, Uppsala, Sweden). Detection, quantification of spots and background subtraction were done for each scanned image. Only those spots which were detected in all gels were selected for
profile analysis. Gels for biological replicates in each group were aligned using landmarks and auto matched. Similarly, gels in different groups (vehicle-treated and onapristone-treated) were also aligned, auto matched and compared for protein expression profile after normalization. Total protein load on each gel was normalized using Image Master 2D Platinum software to take into account the minor variations in sample load, gel staining and destaining. For each spot relative spot volume was calculated by dividing spot volume value by the sum of total spot volume values. Total spot volume was calculated by the software and this referred to the sum volume of all spots on a gel. The volume of each spot was normalized as relative volume in the gels run for all biological replicates in each group as well as in the gels of the groups to be compared. Each detected spot within each gel was assigned a unique number by the software. Analysed gels were matched to a reference gel, protein groups were established and spot group number was assigned to each spot. Ratio of the relative spot volumes of the members of each group (representing a spot sharing same coordinates in the gels to be compared) was calculated by the software. This generated data on the expression profile or abundance of each protein spot in the group. The protein was considered as differentially expressed if its mean relative volume (mean of at least three replicates) was more than 2-fold higher or lower as compared with its counterpart spot in the group.
Mass spectrometry Gel plugs containing the protein spots of interest were manually excised from 2D gels. Gel plugs were washed four times with sterile water by centrifugation at 13 709 g for 10 min at 48C and then processed for MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) analysis on a Biflex III MALDI-TOF mass spectrometer (Bruker Daltonik, Gmbh, Bremen, Germany) at the Central Instrumentation Facility, Jawaharlal Nehru University, New Delhi, India. Each plug was destained using a 1:1 mixture of 30 mM potassium ferricyanide and 100 mM sodium thiosulfate followed by digestion with 0.01 mg/ml sequencing grade trypsin (Applied Biosystems) for 16 h at 378C. Tryptic peptides were extracted from gel plugs using trifluoroacetic acid (TFA) and dried. The peptides were reconstituted in sample diluent (30% v/v acetonitrile, 0.1% TFA) and mixed with an equal volume of matrix (a-cyano-4-hydroxycinnamic acid, 70% v/v acetonitrile, 1% TFA), spotted in duplicate on a target plate and air-dried. The MALDI-TOF was operated in the positive ion delayed extraction reflector mode for highest resolution and mass accuracy. Peptides were ionized with 355 nm laser and spectra were acquired at 20 kV acceleration potential with optimized parameters. Most of the observed peptide peaks of trypsin autolysis and keratin were excluded from monoisotopic precursor ion list generated for tandem mass spectrometry (MS/MS analysis). A maximum of the 25 strongest precursor ions per sample were selected for MS/MS analysis. In the TOF1 stage, all ions were accelerated to 1 kV under conditions, which facilitated fragmentation. The peak detection criteria used were S/N of 10 and local noise window width of 200 (m/z). Combined MS and MS/MS spectra were used to search against the MSDB database (an integrated database of several primary databases i.e. PIR1, PIR2, PIR3, PIR4, TrEMBL, GenBank and Swissprot) to determine the protein identity using BioTools (Bruker Daltonik) or the GPS software (version 3.5, Applied Biosystems) and mascot search algorithm (Matrix Science, London, UK) for peptide and protein identification. Searches were carried out to allow for carbamidomethylation (C), oxidation (M), trypsin as an enzyme and maximum of 1 missed trypsin cleavage. A mass tolerance of 100 ppm and 0.25 Da was used for precursors and fragment ions respectively. The peptide mass fingerprinting (PMF) of the proteins were scored with the Mowse score. Mowse score has been defined as 210 log (P) where P is the probability that the match is a
2208 random event. The criteria used for a positive identification was a significant Mowse score (P , 0.05). Mowse score 64 was considered significant (P , 0.05). If more than one protein met the threshold criteria, the entry with the highest Mowse score was assigned.
Immunohistochemical analysis Endometrial sections were deparaffinized in xylene and rehydrated through descending grades of alcohol. Endogenous peroxidase activity was quenched by incubating with 0.3% H2O2 for 30 min. The tissues were blocked with 1.5% normal swine or horse serum in phosphatebuffered saline (PBS), pH 7.4 for 30 min and then incubated at 48C for 16 h with respective primary antibodies [1:200 dilution of mouse monoclonal PDI antibody (Affinity Bioreagents) or rabbit polyclonal calreticulin or endoplasmin antibody (Sigma-Aldrich, USA)]. Specificity of these antibodies was checked by western blot analysis. These antibodies detected single bands in monkey endometrial protein lysates and the molecular sizes of these proteins were same as observed for human endometrial protein lysates (data not shown). Two negative controls were used in each experiment to check the specificity of immunolocalization by the primary antibody. One negative control was incubated with PBS and another with rabbit immunoglobulin, in place of primary antibody against calreticulin or endoplasmin. Negative control sections for PDI were stained with PBS/myeloma supernatant. The next day, sections were washed twice in PBS for 10 min each and incubated for 2 h at room temperature in respective secondary antibodies [1:50 dilution of horse antimouse biotinylated antibody (Vector Laboratories, Burlingham, USA) for detection of PDI and 1:200 dilution of swine anti-rabbit biotinylated antibody (DAKO, Inc. Carpinteria, CA, USA)] for detection of calreticulin and endoplasmin; prepared in respective 1% blocking solution. After washing in PBS, the sections were incubated with avidin: biotinylated horse-radish peroxidase (HRP) complex for 30 min followed by a PBS rinse and incubation in diaminobenzidene (Sigma) and H2O2 in PBS for 10 min. The sections were dehydrated, cleared in xylene, mounted in dibutyl phthalate xylene (DPX) and viewed under the Olympus BX60 microscope. The intensities of immunostaining (as indicated by the density of brown colouration) were quantified by the image analysis software Biovis 1.42. Briefly, five areas from each section were randomly selected for each animal. The integrated optical density (IOD) per unit area was calculated using the software. Negative control slides were analysed in a similar fashion. The IOD values of the respective negative control (without primary antibody) were subtracted from the IOD values for the sections stained with primary antibody. The mean and SD were calculated for each group. Statistical analysis was carried out using the student’s t-test. Tukey– Kramer test was used to compare all pairs of means following one-way analysis of variance for comparison of the intensities of immunostaining for endometrial calreticulin, endoplasmin and PDI in nongestational/ implantation pole/non-implantation pole gestational endometria. Statistical significance was set at P , 0.05.
Immunoblot analysis Proteins (20 mg) separated on 10% SDS-PAGE were transferred to polyvinylidene difluoride membranes (Amersham). Membranes were incubated in blocking solution [2% blotto made in PBST (PBS with 0.05% tween 20)] for 1 h at room temperature and then incubated with primary antibody against calreticulin or b actin (mouse anti-human, Sigma-Aldrich, USA) diluted 1:1000 in blocking reagent for 16 h at 48C. Membranes were washed extensively with PBST for 1 h with several changes. Membranes were incubated with respective secondary antibody [swine anti-rabbit conjugated to HRP from DAKO or horse anti-mouse conjugated to HRP from Vector] at a dilution of 1:10 000 in PBST. Washings with PBST were for 3 h with several changes. Immunoreactive bands on the membranes were detected
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using advanced enhanced chemiluminescence kit (Amersham). Membranes were stripped and probed with antibodies against b actin. Immunoreactive b actin band served as an internal control for the protein load in each sample. Volumes of immunoreactive bands for calreticulin and b actin were measured using GeneTools software (Synoptics Ltd, UK) in the gel documentation system (Syngene, UK).
Real-time PCR analysis Total RNA was extracted from cell line lysates using RNA extraction kit (Qiagen, GmbH, Germany). Complementary DNA (cDNA) was synthesized using HIGH PRIME cDNA synthesis kit and random hexamers from Applied Biosystems. Biplex real-time PCRs were carried out using 6 carboxy fluorescine (FAM) (6 carboxy fluorescein) labelled calreticulin and VIC (patented by Applied Biosystems) labelled 18S rRNA taqman primer probes (Applied Biosystems). 18S rRNA was used as an endogenous control for the RNA input. Relative quantity (RQ) of calreticulin transcripts was determined using RQ Manager software (Applied Biosystems). The difference in calreticulin transcript levels in hormone-stimulated cultures as compared with unstimulated cultures was estimated by comparing mean RQ + SD (mean of RQs from the experiments at three different time points). Tukey’s multiple comparison test was used to assess the significance of differences in calreticulin transcript levels between various groups.
Results Comparison of 2D protein maps of endometria from vehicle- and antiprogestin-treated bonnet monkeys The peripheral levels of E2 and progesterone did not change significantly after the vehicle or antiprogestin treatment. Figure 1 depicts circulatory levels of E2 and progesterone in pretreatment (A, C) and treatment (B, C) cycles. This reaffirmed our previous observation that the onapristone treatment at 5.0 mg for one cycle does not significantly change the peripheral levels of E2 and progesterone. However, endometrial development was found retarded in antiprogestin treated animals (Fig. 2). Glandular diameter was reduced and there was loss of stromal edema in endometria from antiprogestin treated animals (Fig. 2B, D). These results were also in agreement with our previous observations (Patil et al., 2005). Densitometric analysis of the 2D maps of endometrial tissue proteins (250 mg), resolved in non-linear range of pH 3–11, revealed ca. 668 and ca. 583 polypeptides spots in vehicle-treated control and onapristone-treated animals respectively (Fig. 3A, B). Lesser amount of protein (150 mg) from receptive and non-receptive endometria was separated in the linear range of pH 4–7, first by IEF and then by 10% denaturing SDS-PAGE (Fig. 3C, D); to resolve the spots differing by very small units of pH. Silver stained 2D gels, when analysed by Image Master software, demonstrated ca. 267 and ca. 150 polypeptide spots in vehicle-treated and onapristonetreated animals, respectively. Sixty-five polypeptide spots were found to be differentially abundant in control and antiprogestin-treated animals. Densitometric intensities of 17 protein spots were found to be higher and of 48 protein spots, lower by at least 2-fold in the endometria of vehicle-treated animals as compared with that of onapristone-treated animals. These observations indicated that suboptimal progesterone action due to antiprogestin treatment alters
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Figure 1 Serum estradiol (open circles) and progesterone (dark circles) levels across the pretreatment (A, C) and treatment (B, D) cycles in bonnet monkeys, representing two groups, one treated with vehicle alone (n ¼ 3) and another with the antiprogestin, onapristone (n ¼ 3). Steroid levels were estimated by specific radioimmunoassays. Black bars indicate duration of the menses.
Identification of reticuloplasmins as differentially expressed proteins PMF spectra as well as MS/MS analysis identified two proteins which were more abundant in the 2D endometrial map of antiprogestin-treated animals as compared with that of vehicle-treated animals. These proteins, which migrated at approximately 60 and 56 kDa in the 2D gels, were identified as calreticulin (pI-4.1) and PDI (pI-5.8), respectively (Table I). Mowse scores for both proteins were significant (P , 0.05). Interestingly, the molecular mass of calreticulin, which migrated at 60 kDa in the gels, is estimated to be of 46 kDa on the basis of its amino acid sequence. It has been proposed that highly charged C terminal domain and acidity (pI-4.1) is responsible for the aberrant migration of calreticulin in gels (Fliegel et al., 1989).
Figure 2 Histomorphological features of receptive endometria from vehicle-treated (receptive) (A, C) and non-receptive endometria (B, D) from onapristone-treated bonnet monkeys. Endometria were collected from three vehicle-treated and three onapristone-treated animals. Magnification-10 for A, B and 40 for C, D.
the expression levels of several endometrial proteins. Two proteins which demonstrated a 2-fold increase in their spot volumes in the 2D maps of endometria from antiprogestin-treated animals (Fig. 3D), were analysed for their PMF/MS–MS spectra.
Immunohistochemical localization of calreticulin, PDI and endoplasmin in the functionalis region of endometria of vehicleand antiprogestin-treated bonnet monkeys Immunoreactive calreticulin was localized in cytoplasm and perinuclear region of glandular epithelial cells. Calreticulin localization was predominantly perinuclear and nuclear in some stromal cells. When endometrial functionalis of antiprogestin-treated animals was compared with that of vehicle-treated animals, calreticulin level was significantly higher (P , 0.016) in the glands and lower (P , 0.005) in the stroma of antiprogestin-treated (non-receptive) animals (Fig. 4A-a, e and B). It may be concluded that the higher representation of calreticulin seen in the 2D maps of endometria from antiprogestin-treated
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Figure 3 Two dimensional (2D) protein maps of endometrial proteins from vehicle-treated control (A, C) and onapristone-treated bonnet monkeys (B, D), resolved either in non-linear pH range of 3 – 11 (A, B) or in linear range of 4 – 7 (C, D) by isoelectric focussing in first dimension and 10% SDS-PAGE in second dimension. Total proteins (250 mg) were loaded in 3 – 11 IPG strips and 150 mg in 4 – 7 IPG strips for focussing. Gels were run thrice for each sample (vehicletreated control and antiprogestin-treated groups had three biological replicates each). The maps presented here are of the gels representing spots consistently seen in all three runs. Only those spots, which were seen in all three animals of each group were analysed for differential expression. Differentially abundant spots were identified in the gels (control versus antiprogestin-treated samples), normalized for their total spot volume by imagemaster software. Arrows represent proteins which were up-regulated either in the receptive or non-receptive endometria, as compared with the other. Locations of calreticulin (Cal) and protein disulphide isomerose (PDI) in the 2D protein map are indicated in the panel D.
Table I Mass spectrometry analysis of spots excised from 2D SDS-PAGE gels Protein Identity
Accession No
Molecular weight (kDa) of the matched protein entry
Isoelectric point (pI) of the matched protein entry
Mowse Score
Peptide Sequence
............................................................................................................................................................................................. Calreticulin
Q9UDG2
46
4.1
119
EQFLDGDGWTSR
Protein disulfide-isomerase (EC 5.3.4.1) ER60 precursor-human
JC5704
56
5.8
69
ELSDFISYLQR; LAPEYEAAATRLK; GFPTIYFSPANKK; YGVSGYPTLKIFR
animals results from the increase of calreticulin in the epithelial compartment. PDI was localized in cytoplasm and nucleus of glands and stroma (Fig. 4A-b, f). When compared with vehicle-treated (receptive) animals, PDI expression in stroma was significantly higher (P , 0.0001) in antiprogestin-treated animals (Fig. 4B). The glandular epithelial compartment did not show significant change in the expression of PDI after antiprogestin treatment (Fig. 4A-b, f).
Endoplasmin demonstrated a similar pattern of localization as shown by calreticulin (Fig. 4A-c, g). However, unlike calreticulin and PDI, endoplasmin expression was not up-regulated in any of the endometrial compartments of antiprogestin-treated animals. In fact, the expression of PDI appeared to be lower in the stroma of non-receptive endometria as compared with receptive endometria, although the difference did not reach significance.
Expression of calreticulin in primate endometrium
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Figure 4 (A) Immunohistochemical localization of endometrial the reticuloplasmins calreticulin (a, e), protein disulphide isomerose (PDI) (b, f) and endoplasmin (c, g) in receptive (vehicle-treated control animals) and non-receptive endometria (onapristone-treated animals). Endometria were obtained from three control (a, b, c) and three onapristone-treated animals (e, f and g) on day 8 post E2 peak. In negative control sections (d), rabbit immunoglobulin (Ig) was used in place of the primary antibody for calreticulin or endoplasmin. In negative control (h), myeloma supernatant was used at the concentration equivalent to the working concentration of PDI. Magnification-40. (B) Semi quantitative analysis of immunoreactive reticuloplasmins in glandular (I) and stromal (II) compartments of receptive (n ¼ 3, vehicle-treated) and non-receptive endometria (n ¼ 3, onapristone-treated) from bonnet monkey. Values are mean with SD. Student’s t-test was used to determine the significance of differences in the intensities of immunostained calreticulin/PDI/. * Indicates statistical significance at P , 0.05.
Immunohistochemical localization of endometrial calreticulin, PDI and endoplasmin in pregnant and non-pregnant bonnet monkeys We assessed the effect of embryonic stimuli on reticuloplasmin expression in adequately progesterone-primed endometrium by immunohistochemistry analysis of sections of uteri from pregnant and non-pregnant animals. Calreticulin expression was significantly higher in both-stroma (P , 0.0001) and glands (P , 0.001) of the IPE (Fig. 5A-d) from pregnant animals as compared with the NGE from non-pregnant animals (Fig 5A-a). Calreticulin expression in IPE was significantly higher (P , 0.01) as compared with NIPE also (Fig. 5B). NIPE had significantly higher (P , 0.05) expression of calreticulin in glandular epithelium as compared with NGE (Fig 5B). However, stromal compartments of NGE and NIPE did not differ significantly in their calreticulin levels. This clearly implied a role of embryonic stimuli in modulating the expression of endometrial calreticulin. These observations also suggest that pregnancy-induced modulation in the expression of calreticulin, though not restricted to IPE, was more pronounced in those regions physically proximal to the embryo or embryonic stimuli. Stromal compartment of the IPE from pregnant animals showed significantly higher expression of PDI (P , 0.001) and endoplasmin
(P , 0.01) as compared with the NGE from non-pregnant animals (Fig. 5A-b, e, c, f), respectively. Stromal compartment of the NIPE from pregnant animals also had significantly higher intensities of immunoreactive PDI (P , 0.01) and endoplasmin (P , 0.05) as compared with the NGE from non-pregnant animals (Fig. 5B-II). However, unlike calreticulin, immunoreactive PDI and endoplasmin levels in stroma did not differ significantly between IPE and NIPE in pregnant animals, thereby suggesting that both uterine poles –implantation and non-implantation poles –respond similarly to the embryonic stimuli that modulate PDI and endoplasmin expression. Further in contrast to calreticulin, glandular compartment did not show significant alteration in the levels of immunoreactive PDI and endoplasmin during pregnancy. Interestingly it was the stromal compartment of endometrial functionalis that showed significant increase in the expression of all three reticuloplasmins-calreticulin, PDI and endoplasmin in pregnant animals as compared with non-pregnant animals.
Calreticulin expression in the proliferative and mid-secretory phase human endometria Immunoblot analysis revealed that the mean volume ratio of immunoreactive bands was 1.4-fold higher for calreticulin/b actin in proliferative phase as compared with that in mid-secretory phase human
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Figure 5 (A) Immunohistochemical localization of the reticuloplasmins calreticulin (a, d, g), protein disulphide isomerose (PDI) (b, e, h) and endoplasmin (c, f, i) in pregnant and non-pregnant bonnet monkeys. Gestational (d, e, f, g, h, i) and non-gestational endometria—(NGE, a, b, c) were collected from pregnant (n ¼ 3) and non-pregnant animals (n ¼ 3) on day 9 post-E2 peak. In pregnant animals, endometria from the uterine pole that demonstrated the presence of an embryo are referred as implantation pole endometria (IPE, d, e, f) while endometria from the opposite pole (g, h, i) are referred as non-implantation pole endometria (NIPE). Negative controls (not shown) treated with rabbit Ig/myeloma supernatant and stained with the respective secondary antibodies did not show any detectable staining. (B) Semi quantitative analysis of immunoreactive reticuloplasmin localization in glandular (I) and stromal (II) compartments of gestational and non-gestational endometria. One-way analysis of variance (ANOVA) followed by Tukey–Kramer test was used to compare mean + SD of the IOD values of each group with that of the other group. * And W indicate significant differences in immunostaining intensities between IPE and NGE, or IPE and NIPE, respectively, at P , 0.05.
endometria (Fig. 6). This indicated that the expression of endometrial calreticulin is higher in the estrogen dominant phase (proliferative phase) than in the progesterone dominant (mid-secretory phase) phase of cycle.
In vitro regulation of calreticulin expression in an endometrial cell line Immunoblot analysis of in vitro stimulated Ishikawa cell line revealed up-regulation of calreticulin expression by estrogen and
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Discussion
Figure 6 Immunoblot analysis to determine the relative expression of calreticulin in human endometria collected either during proliferative (P1, P2, P3) or mid-secretory phase (L1, L2, L3). Immunoblots with 20 mg proteins were probed with antibodies against calreticulin. Blots were stripped and reprobed with antibodies against the housekeeping gene beta actin.
Figure 7 Relative quantity (RQ) of calreticulin transcripts in Ishikawa cells stimulated with estradiol or progesterone as compared with unstimulated cultures, as assessed by real-time PCR. Hormone stimulation experiments were repeated at three different time points and RQs were calculated for each set of experiment. RQ of calreticulin transcripts in stimulated cultures as compared with unstimulated cultures was calculated using RQ ¼ 22DDCt where DDCt is the difference between DCts of stimulated and unstimulated Ishikawa cells. DCt for stimulated or unstimulated cultures was calculated by subtracting the Ct (threshold cycle) for 18S rRNA from the Ct for calreticulin. 18S rRNA was used as internal loading or endogenous control for RNA input. * Indicates significant difference (P , 0.05) in the RQs of calreticulin between groups, as calculated by one way ANOVA followed by Tukey Kramer test.
down-regulation by progesterone (data not shown). The mean volume ratio of immunoreactive calreticulin/b actin was 2.8 + 0.9 for estrogenstimulated cultures. These results were further confirmed by real-time PCR studies (Fig. 7). The mean RQ of calreticulin transcripts in cells stimulated with E2 was significantly higher (P , 0.05) as compared with that in vehicle-stimulated and progesterone-stimulated cells. Although calreticulin transcript levels were lower in progesteronestimulated cultures as compared with that in the vehicle-stimulated cells, the difference did not reach statistical significance.
Recently there has been a surge in the microarray-based investigations to identify the genes that are differentially expressed during the progesterone dominant, or mid-secretory, phase as compared with other phases of menstrual cycle in human endometrium (Carson et al., 2002; Kao et al., 2002; Borthwick et al., 2003; Riesewijk et al., 2003; Ponnampalam et al., 2004; Talbi et al., 2006). These studies have provided some clues on putative progesterone-regulated genes. However, attempts to enlist the genes displaying the similar pattern of transcription across all these studies return an extremely low number of genes. This can be attributed to different experimental strategies adopted in these investigations. Further, it is not yet established whether the differential transcription of these putative progesterone regulated genes, translates into the alterations at protein level. On the other hand, it is also possible that some genes may not display any change in their steady state transcript levels, but are subject to post-transcriptional or post-translational regulation. 2D protein maps of endometria from control and antiprogestintreated animals demonstrated ca. 267 and ca. 150 polypeptide spots respectively, in the pH range of 4–7. Imagemaster platinum software analysis revealed differential expression of 65 polypeptides. Forty-eight proteins were up-regulated while 17 were down-regulated in antiprogestin treated animals as compared with control animals. This implied that progesterone induces repression of several genes or gene products to endow the endometrium with receptivity. This is in accordance with a report which demonstrated more genes were down-regulated, than up-regulated in mid-secretory phase, as compared with proliferative phase endometria (Ace and Okulicz, 2004). The positive or negative regulation of proteins by progesterone may occur through direct or indirect mode of progesterone action. Among the differentially expressed proteins, two that showed higher expression in non-receptive endometria as compared with receptive endometria, were identified as calreticulin and PDI. Further it was observed that the increase in the expression of calreticulin and PDI in non-receptive endometria was restricted to specific cell types. For example, increase in calreticulin expression was observed only in glands while increase in PDI expression was seen only in stroma following antiprogestin treatment, implying that the negative regulation of these proteins by progesterone occurs in the context of specific cell type. It may be hypothesized that endometrial glands and stroma differ in their molecular repertoire (i.e. progesterone induced regulators of calreticulin and PDI expression) and hence respond differently to progesterone or antiprogestins. This study is the first to report the differential expression of calreticulin and PDI, both endoplasmic reticulum (ER) resident calciumbinding proteins, or reticuloplasmins, following the blockade of optimal progesterone action on endometrium. ER, a site of synthesis of membrane proteins, membrane lipids and secreted proteins, is also the residence of another calcium-binding protein, endoplasmin or GRP94 (Koch, 1987). Besides contributing to the calcium storage capacity of ER lumen, reticuloplasmins also act as molecular chaperones to prevent the aggregation of partially or incorrectly folded proteins (Michalak et al., 1992). It has been demonstrated that promoter regions of calreticulin, endoplasmin and PDI genes share a number of potential regulatory sites (McCauliffe et al., 1992). These sites include multiple Sp1 and CCAAT consensus sequences, an AP2 recognition
2214 sequence (absent in PD1) and multiple GþC rich areas. This suggests the possibility that calreticulin, endoplasmin and PDI might have similar transcriptional regulation and probably similar function. Since both calreticulin and PDI showed increased expression in endometrium following antiprogestin treatment, we were prompted to investigate the presence and expression pattern of endoplasmin. Contrary to our expectation, endoplasmin expression was not up-regulated in antiprogestin-treated animals. This was an oblique implication that the increase seen in endometrial calreticulin and PDI expression was not a non-specific event induced by antiprogestin treatment and also that despite having similar regulatory genetic elements, all three genes are not co-regulated in endometrium. It was also interesting to observe spatial differences in the localization of calreticulin. In the glands, calreticulin was localized in cytoplasm and perinuclear region whereas in stroma, calreticulin was detected in nucleus and perinuclear region. It will be indeed interesting to investigate whether calreticulin has different modes of action in different cell types, for example genomic mode (transcriptional regulation etc.) in stroma and non-genomic mode (calcium homeostasis and protein folding etc.) in epithelial compartment. In this context, nuclear localization of calreticulin, known to be an ER resident protein, was not surprising. There is evidence to suggest nuclear localization of calreticulin and also the presence of a nuclear localization signal in calreticulin sequence (Opas et al., 1991). Further there are reports that demonstrate binding of calreticulin to the DNA-binding domain of steroid receptors and transcription factors containing the amino acid sequence (KXFFR). It has also been demonstrated that calreticulin prevents the transcription of steroid responsive genes in vitro (Burns et al., 1994). Transcriptional activation by glucocorticoid, androgen, retinoic acid and vitamin D receptors in vivo is modulated in cells over-expressing calreticulin (Michalak et al., 1996; Wang et al., 1997). It has also been proposed that up-regulation in the expression of calreticulin may correlate with increased resistance to steroids (Michalak et al., 1996), raising the possibility that this could be the mechanism by which enhanced expression of calreticulin contributes to endometrial non-receptivity in antiprogestin-treated animals. However, at present, we have no evidence to suggest whether calreticulin over-expression contributes to endometrial nonreceptivity or it is a cellular response to cope with the possible changes in protein folding/assembly/calcium homeostasis in an altered hormonal milieu: considering that these proteins were also highly expressed in endometrium during embryo attachment, the second possibility appears more appealing. Interestingly the levels of GRP78, another reticuloplasmin were found to be increased in the glandular epithelium during uterine sensitization in mice (Simmons and Kennedy, 2000). It has been postulated by the authors that the increased expression of GRP78 is required to facilitate an increase in protein flux through the ER since there is increased protein synthesis and secretion at this time. Immunoblot studies suggesting higher expression of calreticulin in the proliferative phase endometrium as compared with that in the mid-secretory phase endometrium corroborated our recent observation. Calreticulin was found to be more abundant in the 2D maps of proliferative phase than mid-secretory phase endometria collected from regularly cycling healthy women (Parmar et al., 2008), suggesting the possibility of estrogen mediated regulation of endometrial calreticulin. The promoter region of the calreticulin gene contains AP-2 and
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H4TF-1 recognition sequences, typically found in genes that are active during cellular proliferation (reviewed in Michalak et al., 1992). This and another observation demonstrating elevated levels of calreticulin in rapidly dividing cells (Opas et al., 1991) lend support to the possibility of calreticulin being an estrogen-regulated gene. Support for this also came from our in vitro studies, demonstrating an estrogen-induced increase in calreticulin expression in Ishikawa cells. This probably also explains the up-regulation in expression of endometrial calreticulin in antiprogestin-treated bonnet monkeys as well as in pregnant animals. Unopposed estrogen action may be one of the plausible causes of higher expression of calreticulin in the endometrium of antiprogestin-treated animals. In pregnant animals also, estrogenic influences may lead to up-regulation in the expression of calreticulin. In this context, it may be added here that there is an increased expression of endometrial estrogen receptor alpha, an estrogen regulated gene, during early pregnancy (Rosario et al., 2008), indicating that estrogenic influences are operative during initial stages of pregnancy. Data are emerging to suggest that calreticulin acts as a general recognition ligand on apoptotic cells (Gardai et al., 2005; Lim et al., 2008). However, we believe that the increased calreticulin in endometrium during embryo attachment may not signify the onset of apoptosis. First, because during the initial stages of pregnancy (when the embryo attachment ensues), stromal cells do not undergo massive apoptosis, but rather they undergo proliferation followed by differentiation so as to facilitate decidualization. Second, calreticulin localization in perinuclear or nuclear region in endometrium does not concur well with its proapoptotic role reported in other cell types. Further, our studies demonstrating (i) higher expression of endometrial calreticulin in the proliferative phase than in the mid-secretory phase in vivo in humans and (ii) E2 mediated up-regulation in the calreticulin expression, do not support the possibility of calreticulin serving as a signal for apoptosis in endometrial cells. Different experimental strategies were used in this study to investigate the in vivo or in vitro effect of steroids on calreticulin expression in endometrium. One of these strategies was treatment of bonnet monkeys with a select dose of antiprogestin so as to maintain the optimal levels of circulatory progesterone but block the progesterone action on endometrium. On the other hand, the effects of varying levels of progesterone or estrogen on the expression of endometrial calreticulin during normal physiological conditions were assessed using proliferative phase and mid-secretory phase human endometrial biopsies. In vitro studies using Ishikawa cell line helped to determine whether there was any direct effect of hormone supplementation on calreticulin expression. All three strategies revealed that calreticulin expression is lower during ‘progesterone dominance’ than ‘estrogen dominance’ states. To date, there are no reports on the presence or function of reticuloplasmins in primate endometrium. Our study is the first to demonstrate the presence and in vivo hormonal regulation of calreticulin in primates. The study also points out that the response of different endometrial compartments (stroma and glandular epithelium) depends on the type of physiological stimulus. For example, antiprogestin treatment led to increased expression of glandular calreticulin but decreased expression of stromal calreticulin, whereas during pregnancy both compartments showed an increase in calreticulin expression. The study also suggests that despite having similar
Expression of calreticulin in primate endometrium
regulatory elements in their cognate genes, expression of all three reticuloplasmins are not always regulated in identical fashion. Although during embryo attachment a significant increase was observed in expression of these reticuloplasmins in the stromal compartment of endometrial functionalis, antiprogestin treatment altered the expression of calreticulin and PDI but not of endoplasmin. The present study raises several queries, such as (1) whether the increase in calreticulin expression prevents or delays differentiation or maturation of glandular epithelial cells in antiprogestin treated animals? (2) whether the increase in calreticulin expression in glands and stroma in endometrium during embryo attachment promotes stromal and epithelial cell proliferation and thereby decidualization? [our previous studies demonstrated stromal compaction and glandular epithelial cell hyperproliferative activity during early stages of pregnancy (Rosario et al., 2005a, 2008)] and (3) whether increase in the PDI expression in stroma after antiprogestin treatment or during embryo attachment modulates estrogen function? It has been proposed that PDI alters estrogen receptor alpha conformation and influences its ability to mediate changes in gene expression in MCF-7 cells (Schultz-Norton et al., 2006). It will be interesting to investigate whether estrogen receptor mediated gene transcription is altered as a result of increased PDI expression during pregnancy or following antiprogestin treatment. Further investigations are warranted to address these queries and glean information on functional relevance of the differential expression of reticuloplasmins in endometrium under different physiological conditions.
Acknowledgement We would like to thank Dr Surekha Zingade, Deputy Director, Advanced Centre for Training, Research and Education in Cancer (ACTREC), Kharghar, India and Dr Vrinda Khole, Officer-in-Charge and Head, Proteomics Facility NIRRH, India for extending their proteomics facilities. The authors also thank Dr Krishnamurthy Natarajan, Jawaharlal Nehru University, New Delhi and Dr Shalmali Dharma, Proteomics facility, NIRRH for their valuable help in mass spectrometric analysis and peptide sequencing. T.P. was supported by fellowships from the Indian Council of Medical Research. S.N.J. acknowledges the fellowship grant from the Lady Tata Memorial Trust.
Funding Indian Council of Medical Research’s Genomics Grant and the Grant from Department of Science and Technology, Government of India.
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